Biomechanical properties of endothelial cells (ECs) are crucially important in regulation of multiple EC functions, such as mechanotransduction and the integrity of the EC barrier. We have recently discovered that oxidized modifications of LDL (oxLDL) induce significant EC stiffening indicating that dyslipidemia plays a major role in the regulation of EC mechanics. Our long term goal is to elucidate the mechanisms responsible for dyslipidemia-induced changes in EC biomechanics and to determine the contribution of these mechanisms to endothelial dysfunction. During the first funding period of this grant, we have provided the first mechanistic insights into oxLDL-induced EC stiffening and demonstrated that it may facilitate the sensitivity of endothelial cells to flow. In the current proposal, we extend thee studies to address three new goals: In Aim 1, we will identify specific oxidized lipids that induce EC stiffening and address the hypothesis that EC stiffening is mediated by the insertion of oxidized lipids into the plasma membranes of endothelial cells and disruption of lipid packing of membrane domains. To achieve this goal, we will perform Mass Spectrometry analysis of oxidized lipids found in both oxLDL complex and in the vascular walls of aortas isolated from dyslipidemic ApoE-/- mice. EC stiffness will be measured using a combination of two biophysical techniques, Microaspiration and Atomic Force Microscopy and lipid packing will be assayed by two-photon microscopy. In Aim 2, we will elucidate the downstream signaling pathways that are responsible for oxLDL-induced EC stiffening focusing on the roles of caveolin and Rho-GTPases. Specifically, we will address a hypothesis that oxLDL/oxidized lipids-induced disruption of cholesterol-rich membrane domains activate a signaling pathway that includes phosphorylation of caveolin-1, activation of Rho-GTPase and its major downstream target, ROCK, with subsequent changes in actin/myosin organization. This hypothesis will be addressed using an array of gain-of-function and loss-of-function mutants of caveolin, Rho and Rac- GTPases and ROCK. In Aim 3, we will determine the impact dyslipidemia-induced EC stiffening on endothelial permeability under different hemodynamic environments in vitro and in vivo. More specifically, first we will test the hypothesis that oxLDL-induced EC stiffening impairs EC barrier and augments an increase in EC permeability under disturbed pro-atherogenic flow environment in vitro. Finally, we will determine whether an increase in EC stiffness correlates with an increase in endothelial permeability in vivo in ApoE-/- mice and determine whether disruption of the endothelial barrier in ApoE-/- mice can be rescued by caveolin-1 deficiency and/or ROCK inhibition.